“Nuevas estrategias heteroespecíficas en la reproducción 2 asistida...
The development of assisted reproductive technologies (ART) in species of the Felidae family has been proposed to guarantee the genetic viability of species that are on the verge of extinction. Sperm cryopreservation research and IVF have been studied in several Felidae species, but the lack of viab...
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| Formato: | Tesis doctoral acceptedVersion |
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Universidad de Buenos Aires. Facultad de Ciencias Veterinarias
2022
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| Acceso en línea: | http://repositoriouba.sisbi.uba.ar/gsdl/cgi-bin/library.cgi?a=d&c=avaposgra&cl=CL1&d=HWA_7527 https://repositoriouba.sisbi.uba.ar/gsdl/collect/avaposgra/index/assoc/HWA_7527.dir/7527.PDF |
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I28-R145-HWA_7527 |
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dspace |
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Universidad de Buenos Aires |
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I-28 |
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R-145 |
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Repositorio Digital de la Universidad de Buenos Aires (UBA) |
| language |
Español |
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spa |
| topic |
Reproducción asistida Assisted reproduction Felinos Criopreservación ADN Fertilización in vitro Ciencias Veterinarias |
| spellingShingle |
Reproducción asistida Assisted reproduction Felinos Criopreservación ADN Fertilización in vitro Ciencias Veterinarias Duque Rodríguez, Matteo “Nuevas estrategias heteroespecíficas en la reproducción 2 asistida... |
| topic_facet |
Reproducción asistida Assisted reproduction Felinos Criopreservación ADN Fertilización in vitro Ciencias Veterinarias |
| description |
The development of assisted reproductive technologies (ART) in species of the Felidae family has been proposed to guarantee the genetic viability of species that are on the verge of extinction. Sperm cryopreservation research and IVF have been studied in several Felidae species, but the lack of viable homologous oocytes makes it difficult to assess the fertile capacity of sperm after cryopreservation. We propose in chapter 2 to evaluate the ability of cryopreserved sperm from Puma concolor (Pc), Leopardus geoffroyi (Lg) and Panthera onca (Po) to induce fertilization of domestic cat oocytes and subsequent embryonic development prior to implantation. In addition, embryo quality was analyzed based on the number of total cells, the expression of pluripotent (Oct4) and early differentiation (Cdx2) genes, DNA fragmentation and the distribution of the OCT4 protein. After ovariectomy, domestic cat (Felis catus, Fc) oocyte cumulus complexes were matured in vitro in 5% CO2 in humidified air at 38.5°C for 22 h and cryopreserved Pc, Lg, and Po spermatozoa were used for heterologous IVF. The straws were thawed by exposing them to air for 10 s and then immersing them in a 37°C water bath for 30 s. The content of the straws was poured into a sterile 1.5 ml microtube preheated to 37°C. The sperm suspension was diluted (1:3 v/v) by slow enhancement (dropwise) of modified Tyrode's solution. For IVF, in vitro matured oocytes were co-incubated with 2×105 366 motile spermatozoa/mL under 5% CO2 at 38.5°C for 18-20 h. After gamete incubation, putative zygotes were cultured in vitro in 50 µl drops of modified Tyrode's medium in 5% CO2, 5% O2, 90% N2. Cleavage was prolonged 48 h after fertilization and the blastocyst stage was evaluated on day 7. The blastocyst rate was found to be higher using Lg sperm compared to the others. No differences were found in the expression of the pluripotent gene; however, the hPc and hLg hybrids show a higher expression of Cdx2 compared to the Fc embryos. No differences were found between the hPo and Fc hybrids in relation to the expression of the Cdx2 gene. Additionally, hPc blastocysts show less apoptosis and positive cells with OCT4 protein than domestic cat and hLg blastocysts. Hybridization in felines is known, however, no report on the characterization of hybrid preimplantation embryos in vitro has yet been published, and it could be a powerful tool to generate knowledge about sperm inheritance in early embryonic development. Heterospecific embryo transfer of an endangered species has been carried out using recipients of related domestic females. The aggregation of an embryo of an endangered species with a tetraploid embryo of the species to be transferred could improve pregnancy development to term. The main objective of Chapter 3 was to analyze embryo aggregation in a domestic cat model using hybrid embryos. For this purpose, we compared the in vitro development of synchronous (Sync) or asynchronous (Async) and asynchronous with tetraploid (Async4n) aggregation of domestic cat IVF embryos. In addition, the quality of the aggregated blastocyst was analyzed by evaluating the total number of cells, the location of cells by mitotracker staining, the expression of the Oct4, Nanog, Sox2, Cdx2 genes, the number of OCT4+ nuclei, and the presence of DNA fragmentation. In addition, developmental rates of Async4n aggregation of domestic cats with hybrid embryos of Leopardus geoffroyi (hLg) were evaluated. Asynchronous aggregation increased the number of blastocyst cells and the number of OCT4+ nuclei compared to nonaggregated diploid (2n) and tetraploid (4n) embryos. Furthermore, blastocysts produced by Async4n aggregation showed reduced rates of DNA fragmentation. No differences were found in the expression of pluripotent genes, except for the expression of Cdx2, which was higher in 4n and aggregated embryos compared to the control group. Interestingly, hybrid embryos derived from Async4n aggregation with domestic cat embryos have similar rates of development to blastocyst compared with the control. Taken together, the results support the use of two-cell fused embryos to generate tetraploid blastomeres and demonstrate that Async4n aggregation generates good quality embryos |
| author2 |
Salamone, Daniel Felipe |
| author_facet |
Salamone, Daniel Felipe Duque Rodríguez, Matteo |
| format |
Tesis doctoral Tesis doctoral acceptedVersion |
| author |
Duque Rodríguez, Matteo |
| author_sort |
Duque Rodríguez, Matteo |
| title |
“Nuevas estrategias heteroespecíficas en la reproducción 2 asistida... |
| title_short |
“Nuevas estrategias heteroespecíficas en la reproducción 2 asistida... |
| title_full |
“Nuevas estrategias heteroespecíficas en la reproducción 2 asistida... |
| title_fullStr |
“Nuevas estrategias heteroespecíficas en la reproducción 2 asistida... |
| title_full_unstemmed |
“Nuevas estrategias heteroespecíficas en la reproducción 2 asistida... |
| title_sort |
“nuevas estrategias heteroespecíficas en la reproducción 2 asistida... |
| publisher |
Universidad de Buenos Aires. Facultad de Ciencias Veterinarias |
| publishDate |
2022 |
| url |
http://repositoriouba.sisbi.uba.ar/gsdl/cgi-bin/library.cgi?a=d&c=avaposgra&cl=CL1&d=HWA_7527 https://repositoriouba.sisbi.uba.ar/gsdl/collect/avaposgra/index/assoc/HWA_7527.dir/7527.PDF |
| work_keys_str_mv |
AT duquerodriguezmatteo nuevasestrategiasheteroespecificasenlareproduccion2asistida AT duquerodriguezmatteo nuevasestrategiasheteroespecificasenlareproduccionasistidafelina |
| _version_ |
1824356465672978432 |
| spelling |
I28-R145-HWA_75272024-10-18 “Nuevas estrategias heteroespecíficas en la reproducción 2 asistida... The development of assisted reproductive technologies (ART) in species of the Felidae family has been proposed to guarantee the genetic viability of species that are on the verge of extinction. Sperm cryopreservation research and IVF have been studied in several Felidae species, but the lack of viable homologous oocytes makes it difficult to assess the fertile capacity of sperm after cryopreservation. We propose in chapter 2 to evaluate the ability of cryopreserved sperm from Puma concolor (Pc), Leopardus geoffroyi (Lg) and Panthera onca (Po) to induce fertilization of domestic cat oocytes and subsequent embryonic development prior to implantation. In addition, embryo quality was analyzed based on the number of total cells, the expression of pluripotent (Oct4) and early differentiation (Cdx2) genes, DNA fragmentation and the distribution of the OCT4 protein. After ovariectomy, domestic cat (Felis catus, Fc) oocyte cumulus complexes were matured in vitro in 5% CO2 in humidified air at 38.5°C for 22 h and cryopreserved Pc, Lg, and Po spermatozoa were used for heterologous IVF. The straws were thawed by exposing them to air for 10 s and then immersing them in a 37°C water bath for 30 s. The content of the straws was poured into a sterile 1.5 ml microtube preheated to 37°C. The sperm suspension was diluted (1:3 v/v) by slow enhancement (dropwise) of modified Tyrode's solution. For IVF, in vitro matured oocytes were co-incubated with 2×105 366 motile spermatozoa/mL under 5% CO2 at 38.5°C for 18-20 h. After gamete incubation, putative zygotes were cultured in vitro in 50 µl drops of modified Tyrode's medium in 5% CO2, 5% O2, 90% N2. Cleavage was prolonged 48 h after fertilization and the blastocyst stage was evaluated on day 7. The blastocyst rate was found to be higher using Lg sperm compared to the others. No differences were found in the expression of the pluripotent gene; however, the hPc and hLg hybrids show a higher expression of Cdx2 compared to the Fc embryos. No differences were found between the hPo and Fc hybrids in relation to the expression of the Cdx2 gene. Additionally, hPc blastocysts show less apoptosis and positive cells with OCT4 protein than domestic cat and hLg blastocysts. Hybridization in felines is known, however, no report on the characterization of hybrid preimplantation embryos in vitro has yet been published, and it could be a powerful tool to generate knowledge about sperm inheritance in early embryonic development. Heterospecific embryo transfer of an endangered species has been carried out using recipients of related domestic females. The aggregation of an embryo of an endangered species with a tetraploid embryo of the species to be transferred could improve pregnancy development to term. The main objective of Chapter 3 was to analyze embryo aggregation in a domestic cat model using hybrid embryos. For this purpose, we compared the in vitro development of synchronous (Sync) or asynchronous (Async) and asynchronous with tetraploid (Async4n) aggregation of domestic cat IVF embryos. In addition, the quality of the aggregated blastocyst was analyzed by evaluating the total number of cells, the location of cells by mitotracker staining, the expression of the Oct4, Nanog, Sox2, Cdx2 genes, the number of OCT4+ nuclei, and the presence of DNA fragmentation. In addition, developmental rates of Async4n aggregation of domestic cats with hybrid embryos of Leopardus geoffroyi (hLg) were evaluated. Asynchronous aggregation increased the number of blastocyst cells and the number of OCT4+ nuclei compared to nonaggregated diploid (2n) and tetraploid (4n) embryos. Furthermore, blastocysts produced by Async4n aggregation showed reduced rates of DNA fragmentation. No differences were found in the expression of pluripotent genes, except for the expression of Cdx2, which was higher in 4n and aggregated embryos compared to the control group. Interestingly, hybrid embryos derived from Async4n aggregation with domestic cat embryos have similar rates of development to blastocyst compared with the control. Taken together, the results support the use of two-cell fused embryos to generate tetraploid blastomeres and demonstrate that Async4n aggregation generates good quality embryos Fil: Duque Rodríguez, Matteo. Universidad de Buenos Aires. Facultad de Ciencias Veterinarias. Buenos Aires, Argentina Salamone, Daniel Felipe Duque Rodríguez, Matteo 2022-10-05 El desarrollo de tecnologías de reproducción asistida (TRA) en especies de la familia Felidae se ha propuesto para garantizar la viabilidad genética de especies que se encuentran al borde de la extinción. La investigación de la criopreservación de espermatozoides y la FIV se han estudiado en varias especies de Félidos, pero la falta de ovocitos homólogos viables dificulta la evaluación de la capacidad de fertilidad de los espermatozoides después de la criopreservación. Proponemos en el capítulo 2 evaluar la capacidad de los espermatozoides criopreservados de Puma concolor (Pc), Leopardus geoffroyi (Lg) y Panthera onca (Po) para inducir la fecundación de ovocitos de gatos domésticos y el posterior desarrollo embrionario previo a la implantación. Además, se analizó la calidad embrionaria a partir del número de células totales, la expresión de genes pluripotentes (Oct4) y de diferenciación temprana (Cdx2), la fragmentación del ADN y la distribución de la proteína OCT4. Después de la ovariectomía, los complejos cumulus ovocito de gato doméstico (Felis catus, Fc) se maduraron in vitro en 5% de CO2 en aire humidificado a 38.5°C durante 22 h y se usaron espermatozoides criopreservados de Pc, Lg y Po para la FIV heteróloga. Las pajuelas se descongelaron exponiéndolas al aire durante 10 s y luego sumergiéndolas en un baño de agua a 37°C durante 30 s. El contenido de las pajuelas se vertió en un microtubo estéril de 1,5 ml precalentado a 37°C. La suspensión de esperma se diluyó (1:3 v/v) mediante la adición lenta (gota a gota) de una solución de Tyrode modificada. Para la FIV, los ovocitos madurados\nin vitro se incubaron juntamente con 2×105 espermatozoides móviles/mL bajo 5% de CO2 a 38.5°C durante 18-20 h. Después de la incubación de los gametos, los supuestos cigotos se cultivaron in vitro en gotas de 50 µl de medio Tyrode modificado en 5% de CO2, 5% de O2, 90% de N2. El clivaje se determinó 48 h después de la fertilización y el estadio de blastocisto se evaluó el día 7. Se encontró que la tasa de blastocistos fue más alta usando espermatozoides de Lg en\ncomparación con los otros grupos. No se encontraron diferencias en la expresión del gen de pluripotencialidad; sin embargo, los híbridos hPc y hLg presentan una mayor expresión de Cdx2 en comparación con los embriones Fc. No se encontraron diferencias entre los híbridos hPo y Fc en relación con la expresión del gen dx2.Adicionalmente, Los blastocistos hPc presentan menor apoptosis y células positivas con la proteína OCT4 que los blastocistos de gato doméstico y de hLg. Se conoce la hibridación en felinos, sin embargo, aún no se ha publicado ningún informe sobre la caracterización de embriones preimplantacionales híbridos in vitro, y podría ser una herramienta poderosa para generar conocimiento sobre la herencia espermática en el desarrollo embrionario temprano. La transferencia de embriones heteroespecíficos de una especie en peligro de extinción se ha llevado a cabo utilizando receptoras de hembras domésticas emparentadas. La agregación de un embrión de una especie en peligro de extinción con un embrión tetraploide de la especie a transferir podría mejorar el desarrollo de la preñez a término. El objetivo principal del capítulo 3 fue analizar la agregación de embriones en un modelo de gato doméstico utilizando embriones híbridos. Para este propósito, comparamos el desarrollo in vitro de agregación sincrónica (Sync) o asincrónica (Async) y asincrónica con tetraploide (Async4n) de embriones de FIV de gato doméstico. Además, la calidad del blastocisto agregado se analizó mediante la evaluación del número total de células, la asignación de células mediante la tinción de mitotrackers, la expresión de los genes Oct4, Nanog, Sox2, Cdx2, el número de núcleos OCT4+ y la presencia de fragmentación del ADN. Además, se evaluaron las tasas de desarrollo de la agregación Async4n de gatos domésticos con embriones híbridos de Leopardus geoffroyi (hLg). La agregación asíncrona aumentó el número de células de blastocisto y el número de núcleos OCT4+ en comparación con los embriones diploides (2n) y tetraploides (4n) no agregados. Además, los blastocistos producidos por la agregación Async4n mostraron tasas reducidas de ADN fragmentado. No se encontraron diferencias en la expresión de los genes pluripotentes, a excepción de la expresión de Cdx2, que fue mayor en los embriones 4n y agregados en comparación con el grupo control. Curiosamente, los embriones híbridos derivados de la agregación Async4n con embriones de gatos domésticos tenían tasas similares de desarrollo hasta blastocisto en comparación con el control. En conjunto, los hallazgos respaldan el uso de embriones fusionados de dos células para generar blastómeros tetraploides y demuestran que la agregación Async4n genera embriones de buena calidad application/pdf Reproducción asistida Assisted reproduction spa Universidad de Buenos Aires. Facultad de Ciencias Veterinarias info:eu-repo/semantics/openAccess https://creativecommons.org/licenses/by-ncnd/2.5/ar/ Felinos Criopreservación ADN Fertilización in vitro Ciencias Veterinarias Doctor de la Universidad de Buenos Aires en Ciencias Veterinarias Nuevas estrategias heteroespecíficas en la reproducción asistida felina info:eu-repo/semantics/doctoralThesis info:ar-repo/semantics/tesis doctoral info:eu-repo/semantics/acceptedVersion http://repositoriouba.sisbi.uba.ar/gsdl/cgi-bin/library.cgi?a=d&c=avaposgra&cl=CL1&d=HWA_7527 https://repositoriouba.sisbi.uba.ar/gsdl/collect/avaposgra/index/assoc/HWA_7527.dir/7527.PDF |